This project will investigate the mechanisms and pathways for the trafficking of surfactant protein-A (SP-A) and surfactant phospholipid (phosphatidylcholine, PC) by the lung and granular pneumocytes (alveolar epithelial type 2 cells). We hypothesize that: (a) SP-A is taken up by receptor-mediated endocytosis through clathrincoated pits. Endocytosis is facilitated by the novel SP-A binding protein P63 (ERGIC 63);(b) upon stimulation with secretagogues or exposure to SP-A, P63 exits the endoplasmic reticulum compartment, possibly in conjunction with the protein 14-3-3, and travels to the plasma membranes;(c) SP-A reaches lamellar bodies both directly from the endoplasmic reticulum and through an endocytic route;(d) surfactant phospholipid (PC) uptake occurs through at least two different pathways, one clathrin-mediated as a complex with SP-A via receptors and the other clathrin-independent, possibly via: (1) specific lipid binding sites on the type 2 cell membrane such as the lamellar body limiting membranes which have been inserted during the process of exocytosis or (2) lipid rafts. We propose that SP-A plays a role in but is not required for maintenance of normal rates of uptake of surfactant PC under basal condition, since a clathrin-independent pathway can be up-regulated in the absence of SP-A. However, lipid uptake cannot be stimulated above basal levels in SP-Adeficient cells. Studies will be carried out with intact rodents, the isolated perfused lung, and primary cultures of type 2 cells. Specific Aim 1 will examine the interactions of SP-A with the type 2 cell surface membrane protein, P63. Specific Aim 2 will determine intracellular localization and trafficking of P63. Specific Aim 3 will use biochemical techniques to establish the pathway for incorporation of SP-A into lamellar bodies via either direct transfer from the endoplasmic reticulum or endocytosis. Specific Aim 4 will evaluate the clathrindependent and independent pathways for uptake of surfactant PC by type 2 cells using inhibitors specific for each route with SP-A present (wild type) or absent (SP-A KO mouse). These studies will provide evidence defining the pathways regulating the uptake of surfactant components from the alveolar space by the lung and type 2 alveolar pneumocytes.